Increased antimicrobial resistance among Human Immunodeficiency Virus (HIV)-infected individuals to commonly used antibiotics in the treatment of gastroenteritis is a public health concern, especially in resource-limited settings. We set out to compare the antimicrobial susceptibility pattern of Escherichia coli (E. coli) isolates from HIV-infected and HIV-uninfected individuals at a tertiary hospital in Lusaka, Zambia. An analytical cross-sectional study was conducted at the University Teaching Hospital from May 2019 to August 2019. Stool samples were screened, and 79 HIV-infected individuals matched by age and sex with 84 HIV-uninfected individuals that presented with E. coli associated gastroenteritis were studied. Demographics were collected from the Laboratory Information System (LIS) and stool samples were collected in a sterile leak-proof container. Samples were cultured and only those where E. coli was isolated were included in the study and tested for antimicrobial susceptibility by the Kirby–Bauer disk diffusion technique. HIV-positive individuals were 3 times (adjusted odds ratio (AOR) = 3.17; 95% CI (1.51, 6.66); p < 0.001) more likely to be resistant to quinolones compared with their HIV-negative counterparts. Similarly, HIV-positive individuals were almost 4 times (AOR = 3.97, 95% CI (1.37, 11.46); p = 0.011) more likely to have multidrug-resistant E. coli compared with those who were HIV-negative. HIV infection was associated with reduced E. coli susceptibility to commonly used antibiotics, and most cases showed resistance.