The present study aimed to investigate the potential virulence factors and antifungal resistance of 31 Candida albicans and 21 non-albicans Candida isolates colonizing preterm neonates. The study also compared the susceptibility results with the in vitroactivity of chlorhexidine in the eradication of Candida colonization. Candida albicansproduced significantly more phospholipase and coagulase than non-albicans Candida, whereas proteinase production was higher in non-albicans Candida. Biofilm production was demonstrated in Candida albicans and non-albicans Candida (P = 0.214). None of the planktonic growth of Candida isolates were resistant to either fluconazole or amphotericin B, whereas 40% and 84% Candida isolates grown as biofilm became resistant to fluconazole and amphotericin B, respectively. Both coagulase and phospholipase production strongly correlated with the resistance of sessile Candidaisolates to amphotericin B (P < 0.001). Whereas both proteinase and phospholipase correlated with the resistance of in vitro Candida biofilms to fluconazole (P < 0.05 and P = 0.001; respectively). Chlorhexidine was comparable to fluconazole towards planktonic and sessile grown Candida isolates. In conclusion, the study demonstrated an association between certain virulence factors and the development of biofilm drug resistance and highlighted the value of chlorhexidine as a promising prospect in the eradication ofCandida colonization.