Aims Accurate identification of dermatophytes is essential for implementing appropriate antifungal treatment and epidemiological analysis. However, the limitations of conventional diagnostics are a frequently discussed topic, and new diagnostic techniques are constantly expanding. In this study, we assess the suitability of conventional diagnostic techniques in comparison to the real‐time PCR assay and MALDI‐TOF MS in detection and identification of dermatophytes. Methods and Results Strains included in this study were obtained from human and animals with symptomatic, and asymptomatic infection. A direct examination revealed that 31·7 and 60·9% of samples from symptomatic patients, and 25·7 and 60% from asymptomatic animals were positive, as shown by light and fluorescence microscopy respectively. In turn, dermatophytes were isolated from 90·2 and 71·4% of these samples. The pan‐dermatophyte primers in real‐time PCR assay facilitated detection in 85·3 and 82·9% of the symptomatic and asymptomatic dermatophytoses respectively. Additionally, species‐specific PCR assays were positive in 70·7 and 37·1% of these samples. The MALDI‐TOF MS analysis yielded positive results consistent with conventional techniques in 97·2 and 72% of symptomatic and asymptomatic infections respectively. Conclusions Our study revealed that there is no universal diagnostic method that would be ideal in each of the cases considered. Nonetheless, conventional techniques are still the most effective and reliable tools for mycological diagnostics. Significance and Impact of the Study Dermatologists and veterinarians have difficulties in making a diagnosis of dermatophytoses based only on observed symptoms of fungal infections, as they mimic symptoms of other dermatoses. In this context, a comparative analysis of the results of diagnostics performed using conventional methods and new technologies are crucial for implementing these pioneer methods into routine laboratory practice.