The increasing prevalence of multidrug-resistant (MDR) and extended-spectrum beta-lactamase (ESBL)-producing E. coli and K. pneumoniae has become a global health concern. This study was carried out to determine whether there were any correlations between the presence of blaSHV, blaTEM, blaCTX-M and blaOXA genes and biofilm formation in ESBL producing E. coli and K. pneumoniae from a tertiary care referral hospital in Dhaka, Bangladesh. One hundred and fifty urine samples were collected from May 2015 to October 2015. Kirby-Bauer disc diffusion method was carried out to determine the antibiotic resistance pattern. Screening and phenotypic confirmatory tests for ESBL production were performed using the double disc synergy test (DDST) and the presence of blaSHV, blaTEM, blaCTX-M and blaOXA genes was confirmed via polymerase chain reaction (PCR) using gene-specific primers. Biofilm formation was assessed using a semi-quantitative method. One hundred and twenty unique bacteria were isolated from 150 urine samples of which 66.7% (80/120) were E. coli and 33.3% (40/120) were K. pneumoniae. DDST showed that 62.5% (50/80) E. coli and 75% (30/40) K. pneumoniae isolates were ESBL producers. Almost all isolates showed resistance to third generation cephalosporin but 100% sensitivity to colistin. PCR analysis confirmed that 36% (18/50) E. coli possessed blaCTX-M gene and 63.3% (19/30) K. pneumoniae were positive for blaCTX-M gene. Among blaTEM, blaSHV, blaOXA and blaCTX-M genes, blaCTX-M gene was most commonly found in both bacterial species. Strains harboring more resistance genes exhibited more biofilm production, indicating a positive correlation between biofilm formation and multidrug resistance. Further studies with a large number of samples are needed to confirm the association between biofilm production and multidrug resistance among ESBL producers.