Biofilms, comprising extracellular polysaccharides, proteins, and DNA, provide structural strength and protective barriers that limit antibiotic penetration and are a major factor in antimicrobial resistance. This resistance complicates treatment of biofilm-related infections, and research has been directed to explore alternatives such as quorum sensing inhibitors, biofilm-disrupting enzymes, and antimicrobial coatings. Fluoroquinolones, especially moxifloxacin, are synthetic broad-spectrum agents with antibacterial activity against Gram-positive and Gram-negative bacteria. They cause cell death by inhibiting bacterial DNA gyrase and topoisomerase IV. Moxifloxacin is used to treat severe infections due to its strong antibacterial efficacy, immunomodulatory effects, and favorable pharmacokinetics. This study examined the influence of sub-minimum inhibitory concentrations (sub-MICs) of moxifloxacin on antibiotic resistance and biofilm production. Findings revealed that Pseudomonas aeruginosa ATCC 27855 and Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 33591 recorded the highest increase in biofilm production - 89.3% and 194.48%, compared to controls. Sub-MIC moxifloxacin influenced antibiotic resistance, particularly among MRSA ATCC 33591, that was increased upon developing resistance against multiple antibiotics when exposed for 24 and 48 h. All Gram-negative bacteria tested under the test, namely E. coli, P. aeruginosa, K. pneumoniae, and A. baumannii, proved to be resistant to different antibiotics such as ampicillin, erythromycin, and azithromycin. Minor time-dependent inhibitory zone increments were observed as well. These findings highlight the importance of subinhibitory antibiotic levels in enabling both biofilm development and antibiotic resistance, highlighting the importance of thorough susceptibility testing in treating multidrug-resistant infections.