Although Candida spp., is a common cause of bloodstream infections and is often associated with high mortality rates, its resistance to antifungal drugs, and the molecular mechanisms involved have been poorly studied in Colombia. Here, 123 bloodstream isolates of Candida spp. were collected. MALDI-TOF MS identification and fluconazole (FLC) susceptibility patterns were assessed on all isolates. Subsequently, sequencing of ERG11, TAC1 or MRR1, and efflux pumps were performed for resistant isolates. Out of 123 clinical strains, C. albicans accounted for 37.4%, followed by C. tropicalis 26.8%, C. parapsilosis 19.5%, C. auris 8.1%, C. glabrata 4.1%, C. krusei 2.4% and C. lusitaniae 1.6%. Resistance to FLC reached 18%. Erg11 amino acid substitutions associated with FLC-resistance (Y132F, K143R or T220L) were found in 58% of 19 FLC-resistant isolates. Furthermore, novel mutations were found in all genes studied. Regarding efflux pumps, 42% of 19 FLC-resistant Candida spp strains showed significant efflux activity. Finally, six of the 19 FLC-resistant isolates neither harbored resistance-associated mutations nor showed efflux pump activity. Although C. albicans remain the most predominant species, non-C. albicans species comprise a high proportion (62.6%). Among FLC-resistant species, C. auris (70%) and C. parapsilosis (25%) displayed the highest percentages of resistance. In 68% of FLC-resistant isolates, a mechanism that could explain their phenotype was found (e.g. mutations, flux pump activity or both). We provide evidence that endemic isolates harbor amino acid substitutions related with resistance to one of the most used molecules in the hospital setting, with Y132F being the most frequently detected one.