High frequency of antibiotic detection in groundwater in informal settlements is attributed to increased usage and improper disposal, thus difficult to identify sources of antibiotic resistance in the environment, worsened by inadequate sanitation facilities and increased population density, particularly in developing-countries. Reliance on groundwater exposes them to pollutants and risk of antibiotic resistance, in addition to experiencing inequities in accessing vital services. Sulfamethoxazole and trimethoprim, used for prophylaxis by HIV/AIDS patients were tested in 49 groundwater sources in Kisumu, Kenya. Only Sulfamethoxazole (SMX) was found, with a detection frequency of 14.3% and concentrations ranging from below limit of quantification (LOQ) to 258.2 ng/L. Trimethoprim (TMP), marketed in combination with sulfamethoxazole, was not detected, owing to its high distribution coefficient (kPa7.12) and, generally, being a bigger molecule with modest water mobility and solubility. Furthermore, TMP ratio in cotrimoxazole is low (5:1), it is expected that mass loading will be lower, as well as influence of the study area’s hydrogeology, where soil is clayey with high porosity and permeability. Escherichia coli was recovered in 98% (n = 48) of water samples, with counts ranging from 16 to 8,850 MPN/100ml. Additionally, resistance to sulfamethoxazole was identified in 6% (n = 3) samples with Inhibition Zone Diameters of 0.8mm(resistant), 10.5mm (resistant), and 11.5mm (intermediate), but not among samples where SMX was detected. Antibiotic concentrations in water that can cause resistance are unknown because antibiotic-resistant E. coli was not found in water samples where sulfamethoxazole was identified, raising concerns about f environmental resistance spread. Concentration of SMX was lower in a previous research, which only collected water from one groundwater source, than the current study, which included additional samples (49). Presence of SMX and Escherichia coli resistance is of concern and necessitates greater attention and regular monitoring for potential contaminants and resistance trigger to avert potential risks to human health.