Mustard (Brassica juncea L.) is the second most important oilseed commodity in India. Alternaria blight (Alternaria brassicae) is one of the devastating fungal pathogen, resulting in severe yield losses to the crop. The present study is conducted in-vitro to know the effect of different temperature regimes and various culture media on the mycelial growth and sporulation of A. brassicae. It is clearly evident from the results that all the tested temperature regimes and culture media showed variation in the colony diameter and other cultural characteristics. The temperature of 25°C significantly encouraged mycelial growth (85.00 mm) and exhibited excellent sporulation of A. brassicae compared to 15 and 35°C. In case of culture media, Potato Dextrose Agar was found to be significantly superior over the other media and registered maximum mycelial growth (82.00 mm) with excellent sporulation. However, Oat Meal Agar supported the minimum mycelial growth (45.00 mm) with fair sporulation. The variation in cultural characteristics (colony color, growth, appearance, and shape etc.) was also observed with various culture media.