Objective: This study aimed to determine the carriage of extended spectrum β-lactamase (ESBL) genes in third-generation cephalosporin-resistant (3GCR) Escherichia (E.) coli from frequently contacted surfaces, wastewater and disinfectant-cleaning solutions of selected healthcare institutions in South-western Nigeria. Methods: Samples were collected over three months for the isolation of 3GCR E. coli on MacConkey agar containing 6 μg/mL of cefotaxime. 3GCR E. coli isolates were identified by detection of uidA gene and susceptibility to selected antibiotics was performed using disc-diffusion method. Detection of ESBL genes was done using primer-specific PCR. Results: A total of 22 ESBL-producing E. coli (11 each from the frequently contacted surfaces and wastewater) were obtained from the pool of 3GCR isolates in this study. No isolate was recovered from the disinfectant-cleaning solution. All the ESBL-producing E. coli obtained from the frequently contacted surfaces and wastewater were multidrug resistant, with complete resistance observed to ampicillin, cefotaxime, cefpodoxime, tetracycline and ertapenem. The ESBL genotyping showed that 54.5% carried bla_CTX-M, 63.6% carried bla_TEM and 9.1% carried bla_SHV in isolates from the frequently contacted surfaces, while 63.6%, 9.1% and 18.2% carried bla_CTX-M, bla_TEM and bla_SHV, respectively, in the isolates obtained from the wastewater. Conclusions: This study showed a high burden of multidrug resistance E. coli on frequently contacted surfaces and wastewater of the studied healthcare institutions, indicating the need for good hygiene and proper mitigation measures to prevent potential public health and environmental challenges.