An experiment was conducted to find out fungal pathogen associated with leaf samples of Aloe vera obtained from commercial fields of Northern part of Bangladesh. Leaf spot disease causing fungus-Alternaria alternata was identified through morphological characterization based on mycelium, conidia, colony features as well as molecular characterization using internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) of fungi. Molecular identification revealed that ITS sequences of our studied fungus (MH368103.1) was genetically similar to sequences of Alternaria alternata in NCBI database. Typical leaf spot was reproduced by artificial inoculations of the isolated fungus. The mycelial growth of this fungus was evaluated on different culture media i.e., potato dextrose agar (PDA), carrot agar (CA), potato sucrose agar (PSA), Richard agar (RA), Honey peptone agar (HPA), Honey agar (HA); temperature (15, 20, 25, 30, 35⁰C) and light condition (complete light, complete dark, alternate light-dark). A. alternata showed the maximum mycelial growth on PDA at 25⁰C temperature. Both complete dark and alternate light-dark condition were mostly favored for profuse vegetative growth of the fungus. Fungal antagonist-Trichoderma reesei showed very promising results, which inhibited the mycelial growth under in vitro condition. Systemic fungicide Tilt 250EC (500ppm) showed complete inhibition of the vegetative growth of the studied fungus. To the best of our knowledge, Alternaria alternata causing Aloe vera leaf spot disease in Bangladesh, a new record.