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Background Antimicrobial resistance (AMR) is an increasing public health threat in sub-Saharan Africa. Data on the resistance profiles of Escherichia coli in clinical and environmental settings remain limited. This study investigates the prevalence and antimicrobial susceptibility of E. coli in Lusaka to support local antibiotic stewardship and surveillance strategies. Methods A cross-sectional study was conducted from February to June 2023 in Lusaka, Zambia. A total of 450 clinical (urine, blood, wound swabs) and environmental (water, surfaces) samples were collected. Escherichia coli isolates were identified using standard microbiological procedures and confirmed with the VITEK® 2 Compact system. Antimicrobial susceptibility testing was performed according to CLSI 2022 guidelines, and data were analyzed using WHONET 2022 and SPSS v25. Multidrug resistance (MDR) was defined as resistance to at least one antimicrobial agent in at least three antimicrobial categories, and ESBL production was confirmed phenotypically. Results Out of 450 samples, 215 (47.8%) tested positive for Escherichia coli. Clinical isolates accounted for 37.8% of the samples, while environmental samples represented 10%. High resistance rates were observed for ampicillin (81.4%), sulfamethoxazole/trimethoprim (70.7%), ciprofloxacin (67.9%), and ceftriaxone (62.3%). Susceptibility remained preserved for amikacin (100%), meropenem (99.5%), and nitrofurantoin (89.3%). Multidrug resistance (MDR) was detected in 66.5% of isolates, and 42.8% showed ESBL production. The distribution of ESBL-producing strains was comparable between clinical (45.2%) and environmental (38.9%) sources, indicating widespread circulation of highly resistant strains.