Candidiasis refers to both superficial and deep-tissue fungal infections often caused by Candida albicans. The treatment of choice for these infections is the use of azoles, such as fluconazole (FLC). However, the increased use of antifungal agents has led to the emergence of azole-resistant isolates of C. albicans. Thus, the development of alternative drugs that are more efficient and with a better toxicological profile is necessary. This study aimed to determine the susceptibility profile of C. albicans CAF2-1 strain to FLC in the presence of glucose or lactate. The research was also focused on single nucleotide polymorphism (SNP) and the determination of the effect of the identified point mutations on the amino acid sequence of the Erg11 protein. The results show the growth of C. albicans CAF2-1 in the presence of FLC was significantly lower in the presence of lactate than in glucose. As a result, among recorded 45 amino acid mutations, the following mutations may be associated with the reduced susceptibility of C. albicans to FLC: G10D, G10V, I11M, I11R, Y13N, F31V, L35F, A249D, Q250H, E266G, R267G, N273K, D275C, D275G, D275R. Moreover, a twice higher number of hot-spot mutations was found in the presence of glucose as a sole carbon source compared to cells grown on lactate.