Background Escherichia coli (E.coli) rank one of the most common pathogens that can cause neonatal infections. The emergence of antibiotic-resistant bacteria is a major cause of treatment failure in newborn with infection. The purpose of this study was to describe the antibiotic resistance and multidrug-resistance of E.coli isolated from neonates with infection.Methods The antimicrobial susceptibility testing of the E. coli strains to selected antibiotics was assessed with the E-test technique on the Mueller-Hinton agar. The antimicrobials tests were included ceftazidime, cefuroxime, cefatriaxone, amoxicillin, amoxicillin-clavulanic acid, cefoperazone - sulbactam, meropenem, gentamicin, ciprofloxacin and sulfonamides. The minimal inhibitory concerntration (MIC) values of the antimicrobial agents selected for this study was determined by an agar dilution technique on Mueller-Hinton agar according to the Clinical and Laboratory Standards Institute recommendations. Results A total of 100 E. coli strains was isolated from phlegm (n = 78), blood (n = 10), cerebrospinal fluid (n = 5), and umbilical discharge (n = 7) of neonates hospitalized at Beijing Children’s Hospital. The highest resistance rate of E.coli was found in amoxicillin at 85%, followed by cefuroxime 65%, and cefatriaxone 60%, respectively. 6% and 5% of all isolates were resistant to amoxicillin/clavulanic acid and cefoperazone -sulbactam merely. The resistance rates to ceftazidime, gentamicin, ciprofloxacin and sulfonamides were 31%, 20%, 33%, 47%, respectively. All the isolates were susceptible to meropenem. Multidrug resistance was defined in E.coli as resistance to at least three antibiotic families. About 26% (26/100) of all the E.coli isolates were multidrug-resistant. The detection rate of ESBL-Producing E. coli was 55%. The rate in E. coli isolates from phlegm was higher than aseptic humoral. The difference was statistically significant (P < 0.05). It is worth noting that the majority of the isolates were also resistant to non-β-lactam antimicrobial agents, but the resistant rates were significantly lower than extended-spectrum β-lactamases.Conclusions: Multi-drug-resistant E.coli has become a thorny problem in clinical treatment. It is necessary to monitor E. coli resistance.