The die-back of neem caused by Phomopsis azadirachtae is a devastating disease in India reducing the life span and seed production of neem. Six isolates of antagonistic bacteria and fungi, Bacillus cereus (MTCC 430), B. subtilis (MTCC 619), Pseudomonas aeruginosa (MTCC 2581), P. oleovorans (MTCC 617), Trichoderma harzianum (MTCC 792) and T. viride (MTCC 800) were evaluated against P. azadirachtae under in vitro conditions. Culture filtrates of all these microorganisms were extracted using ethyl acetate, and the obtained fractions were tested for their antifungal activity against P. azadirachtae at different concentrations. Ethyl acetate extracts of B. subtilis and P. aeruginosa were highly effective and completely inhibited the growth of P. azadirachtae at 25 ppm concentration. Both these isolates may be considered as factors for the biological control of die-back of neem.