<p>Hospital effluent and domestic sewage contain a complex mix of organic pollutants, heavy metals, and pathogens posing environmental and public health risks. Understanding local toxic chemical pollutants and their effects at low concentrations is important to evaluate how they trigger bacterial stress responses like biofilm formation, contributing to antibiotic resistance. The reported study analyzed the chemical composition of wastewater from different sources (hospital, domestic, and institutional) in Aligarh City, India, and evaluated in vitro toxicity and biofilm development in Gram-negative bacteria. Wastewaters collected during 2022–23 were subjected to routine microbiological and physicochemical analysis using standard methods. Phytotoxicity was assessed in <i>Vigna radiata</i>. Biofilms were examined using a microtiter plate assay and microscopy. Physicochemical and microbiological analysis of wastewater revealed site-specific variability in pH, salinity, major ions, and microbial load/coliforms. Cu was the dominant metal in hospital and sewage water (0.068 – 0.079&#xa0;mg/L⁻¹). GC–MS revealed mainly alkanes, phthalates, fatty acids, and esters. DCM extracts from sewage and hospital sources resulted in phytotoxic effects (up to 75%) on <i>V. radiata</i> and affected bacterial planktonic viability (4.1–8.3 log₁₀CFU/mL) in a concentration-dependent manner. Oxidative damage was notable in <i>V. radiata</i> roots exposed to 25% hospital wastewater. The maximum tolerance concentration (MTC) of heavy metals ranged from 50–800&#xa0;µg/mL. Low metal levels (≤ MTC/8) enhanced biofilms while MTC inhibited biomass, as confirmed microscopically. Hospital/sewage pollutants impaired seed germination and bacterial viability, while sub-toxic metals promoted biofilm formation. Further studies are required to identify pollutants’ role, including antibiotics, in biofilm development and resistance evolution.</p>