Aims:  The aim of this study was to investigate the genetic relatedness between Candida albicans isolates and to assess their nosocomial origin and the likeliness of cross‐transmission between health care workers (HCWs) and hospitalized neonates in a neonatal intensive care unit (NICU). Methods:  We retrospectively analysed 82 isolates obtained from 40 neonates and seven isolates from onychomycosis of the fingers of five HCWs in a Tunisian NICU by using pulsed‐field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) analysis with CA1 and CA2 as primers. Results:  In RAPD analysis, the discriminatory power (DP) of CA1 and CA2 primers was 0·86 and 0·81, respectively. A higher DP was achieved by combining patterns generated by both primers (0·92), while PFGE karyotyping exhibited the lowest DP (0·62). The RAPD‐CA1/CA2 analysis revealed that 65·8% of isolates obtained from neonates derived from a limited number (6) of groups of genetically identical strains, that five temporal clusterings occurred during the study period and that three HCWs’ isolates and 11 isolates obtained from six neonates were identical. Conclusions:  These findings argue for the nosocomial transmission of C. albicans in our NICU and for the transfer of strains from HCWs to patients. Significance and Impact of the Study:  Identification of relatedness between Candida species obtained from neonates and health care workers by using molecular techniques with high discriminatory power is essential for setting up specific control measures in order to reduce the incidence of nosocomial candidiasis.