The recent investigation was designed to explore Scutellaria petiolata Hemsl. ex Lace & Prain (Lamiaceae) whole plant in various extracts (methanol (SPM), dichloromethane (SPDCM), n-Hexane (SPNH), and aqueous (SPAQ) for a phytochemicals assessment, ESI-LC-MS chemical analysis, in vitro antimicrobials, and antioxidants and in vivo anti-inflammatory and analgesic potential. The qualitative detection shows that all the representative groups were present in the analyzed samples. The examined samples display the greatest amount of total flavonoid content (TFC, 78.2 ± 0.22 mg QE/mg) and total phenolic contents (TPC, 66.2 ± 0.33 mg GAE/g) in the SPM extract. The SPM extract proceeded to the ESI-LC-MS to identify the chemical constituents that presented nineteen bioactive ingredients, depicted for the first time from S. petiolata mainly contributed by flavonoids. The analyzed samples produced considerable capability to defy the microbes. The SPM extract was observed effective and offered an appreciable zone of inhibition (ZOI), 17.8 ± 0.04 mm against the bacterial strain Salmonellatyphi and 18.8 ± 0.04 mm against Klebsiella pneumonia. Moreover, the SPM extract also exhibited 19.4 ± 0.01 mm against the bacterial strains Bacillus atrophaeus and 18.8 ± 0.04 mm against Bacillus subtilis in comparison to the standard levofloxacin (Gram-negative) and erythromycin (Gram-positive) bacterial strains that displayed 23.6 ± 0.02 mm and 23.2 ± 0.05 mm ZOI, correspondingly. In addition to that, the SPD fraction was noticed efficiently against the fungal strains used with ZOI 19.07 ± 0.02 mm against Aspergillus parasiticus and 18.87 ± 0.04 mm against the Aspergillus niger as equated to the standard with 21.5 ± 0.02 mm ZOI. In the DPPH (2,2-diphenyl-1-picrylhydrazyl) analysis, the SPM extract had the maximum scavenging capacity with IC50 of 78.75 ± 0.19 µg/mL succeeded by the SPDCM fraction with an IC50 of 140.50 ± 0.20 µg/mL free radicals scavenging potential. Through the ABTS (2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) assay, the similar extract (SPM) presented an IC50 = 85.91 ± 0.24 µg/mL followed by the SPDCM fractions with IC50 = 182.50 ± 0.35 µg/mL, and n-Hexane fractions were reported to be the least active between the tested samples in comparison to ascorbic acid of IC50 = 67.14 ± 0.25 µg/mL for DPPH and IC50 of 69.96 ± 0.18 µg/mL for ABTS assay. In the in vivo activities, the SPM extract was the most effective with 55.14% inhibition as compared to diclofenac sodium with 70.58% inhibition against animals. The same SPM crude extract with 50.88% inhibition had the most analgesic efficacy as compared to aspirin having 62.19% inhibition. Hence, it was assumed from our results that all the tested samples, especially the SPM and SPDCM extracts, have significant capabilities for the investigated activities that could be due to the presence of the bioactive compounds. Further research is needed to isolate the responsible chemical constituents to produce innovative medications.