Background: Beef cattle, one of the food producing animals is linked to humans through shared environment and the food chain as a major source of animal protein. Antimicrobial drugs are readily accessible for use in food animal production in Nigeria. Beef cattle and abattoir environments harbor pathogenic bacteria such as Escherichia coli (E. coli) which have developed resistance to antimicrobial agents used for prophylaxis or treatment. This study aimed to investigate the zoonotic transmission of Extended-spectrum beta lactamase producing E. coli (ESBL-EC) among humans, beef cattle, and abattoir environments in Abuja and Lagos, Nigeria. Methods: We conducted a cross-sectional study among abattoir workers, beef cattle and abattoir environments in Abuja and Lagos. Stool, caecal, and environmental samples were collected from apparently healthy workers, slaughtered cattle, and abattoir environments from July to December 2020. Data were collected electronically using an open data kit (ODK) installed on a smartphone. Antimicrobial susceptibility patterns were determined using Kirby Bauer disk diffusion method against a panel of 16 antimicrobial agents. Phenotypic and genotypic characterization of the isolates was conducted. Data were analyzed with descriptive statistics. Results: Of 448 samples, 21.7% (n=97) were confirmed ESBL producers by phenotypic characterization. Prevalence of ESBL-EC was highest in slaughtered beef cattle (45.4%; n=44), abattoir workers (41.2%; n=40) and abattoir environment (13.4%; n=13). Among ESBL producers, we observed the dissemination of blaCTX-M-15 (90.7%; n=88); blaCTX-M-14 (5.2%; n=5); blaCTX-M-55 (2.1%; n=2); and blaOXA-1 (1.0%; n=1) genes. The blaCTX-M-15 coexisted with blaCTX-M-14 and blaTEM-1 genes in 2.1% (n=2) and 39.2% (n=38) of the isolates respectively. Isolates from all sources showed equal distribution of insertion sequences and miniature inverted repeats although only few composite transposons were detected (Humans n=12; Cattle n=9; Environment n=4). Conclusions: ESBL-EC was detected in apparently healthy abattoir workers and beef cattle and the abattoir environment in two large abattoirs in Nigeria. It is important to educate healthcare workers that people who work closely with cattle are a high-risk group for faecal carriage of ESBL-EC when compared to the general population. Keywords: Escherichia coli, antimicrobial resistance, beef cattle, extended-spectrum β-lactamases (ESBL), Nigeria