This study aimed to assess the markers of chemical and microbiological contamination of the air at sport centers (e.g., the fitness center in Poland) including the determination of particulate matter, CO2, formaldehyde (DustTrak™ DRX Aerosol Monitor; Multi-functional Air Quality Detector), volatile organic compound (VOC) concentration (headspace solid-phase microextraction coupled with gas chromatography–mass spectrometry), the number of microorganisms in the air (culture methods), and microbial biodiversity (high-throughput sequencing on the Illumina platform). Additionally the number of microorganisms and the presence of SARS-CoV-2 (PCR) on the surfaces was determined. Total particle concentration varied between 0.0445 mg m−3 and 0.0841 mg m−3 with the dominance (99.65–99.99%) of the PM2.5 fraction. The CO2 concentration ranged from 800 ppm to 2198 ppm, while the formaldehyde concentration was from 0.005 mg/m3 to 0.049 mg m−3. A total of 84 VOCs were identified in the air collected from the gym. Phenol, D-limonene, toluene, and 2-ethyl-1-hexanol dominated in the air at the tested facilities. The average daily number of bacteria was 7.17 × 102 CFU m−3–1.68 × 103 CFU m−3, while the number of fungi was 3.03 × 103 CFU m−3–7.34 × 103 CFU m−3. In total, 422 genera of bacteria and 408 genera of fungi representing 21 and 11 phyla, respectively, were detected in the gym. The most abundant bacteria and fungi (>1%) that belonged to the second and third groups of health hazards were: Escherichia-Shigella, Corynebacterium, Bacillus, Staphylococcus, Cladosporium, Aspergillus, and Penicillium. In addition, other species that may be allergenic (Epicoccum) or infectious (Acinetobacter, Sphingomonas, Sporobolomyces) were present in the air. Moreover, the SARS-CoV-2 virus was detected on surfaces in the gym. The monitoring proposal for the assessment of the air quality at a sport center includes the following markers: total particle concentration with the PM2.5 fraction, CO2 concentration, VOCs (phenol, toluene, and 2-ethyl-1-hexanol), and the number of bacteria and fungi.