Background: Dermatophytosis is a fungal infection that can be zoonotic, with transmission occurring in both directions between humans and companion animals, particularly in settings involving close contact. This study aimed to determine the prevalence and identify the causative agents of dermatophytosis in dogs and cats using conventional and molecular diagnostic methods. A total of 150 animals with dermatological lesions were sampled, including 105 cats and 45 dogs from both household and shelter environments. This cross-sectional study employed direct microscopy and fungal culture as the initial diagnostic methods. PCR targeting the CHS1 gene was subsequently performed on fungal isolates obtained from 38 culture-positive samples, followed by species-specific amplification to identify Microsporum canis and Trichophyton rubrum. For molecular identification, DNA was extracted from pure cultures derived from hair, skin scrapings, and nail specimens. ITS region sequencing was also performed on two of the PCR-confirmed T. rubrum isolates. Prevalence was compared across animal species, age groups and living environments. Results: Dermatophytes were detected in 25.3% (38/150) of samples. In cats, only M. canis 76% (19/25) was identified. In dogs, both M. canis (5/13) and T. rubrum (2/13) were found. This represents the first report of T. rubrum in a dog in Türkiye, with ITS sequencing confirming > 99% identity to reference strains. Infection rates were significantly higher in animals under one year of age (p = 0.0097), while no statistically significant difference was observed between dogs and cats (p = 0.529). PCR and sequencing provided rapid and accurate identification. Conclusions: Dermatophyte infections are more prevalent among juvenile animals and pose a growing zoonotic threat. Molecular diagnostics improve early detection and control strategies. These findings highlight the need for routine surveillance and reflect the critical importance of the One Health approach, which integrates human, animal, and environmental health to prevent and manage zoonotic disease transmission.