In bacterial infections, including urinary tract infections (UTIs), the gap between the development of new antimicrobials and antimicrobial resistance is dramatically increasing, especially in Gram-negative (Gram–) bacteria. All healthy products that can be used per se or that may be sources of antibacterial compounds should be considered in the fight against this major public health threat. In the present study, the phytochemical composition of Aloe vera extract was investigated by HPLC–MS/MS, and we further evaluated its antibacterial and antibiofilm formation activity against selected resistant Gram– bacteria involved in UTIs, namely, Achromobacter xylosoxidans 4892, Citrobacter freundii 426, Escherichia coli 1449, Klebsiella oxytoca 3003, Moraxella catarrhalis 4222, Morganella morganii 1543, Pseudomonas aeruginosa 3057, and a reference strain E. coli ATCC 25922. Inhibition zones (IZs) of the extract were determined using the well diffusion method, minimum inhibitory (MIC), and bactericidal (MBC) concentration by the two-fold serial microdilution assay, and antibiofilm formation activity by the crystal violet attachment assay. Aloe-emodin and its derivatives were the major constituent (75.74%) of A. vera extract, the most important of them being aloesin (30.22%), aloe-emodin-diglucoside (12.58%), and 2′-p-methoxycoumaroylaloeresin B (9.64%). The minerals found in the extract were sulfur (S), silicon (Si), chlorine (Cl), potassium (K), and bromine (Br). Except for the clinical strain E. coli 1449, which was totally non-susceptible, A. vera demonstrated noteworthy antibacterial activity with MIC and MBC values ranging from 0.625 to 5 mg/mL and 5 to 10 mg/mL, respectively. A. vera also demonstrated dose-dependent antibacterial effects, and the reference strain E. coli ATCC 25922 was the most susceptible with MIC = 0.625 and IZ = 19 mm at 20 mg/mL. The antibiofilm formation potential of A. vera extract was strong at 2MIC and MIC (93–100% of biofilm formation inhibition), moderate at MIC/2 (32–41%), weak at MIC/4 (14–21%), and nil at MIC/8.