Abstract: The aim of this work was to investigate the effect of Antibiotics on germination responses in vitro on mature seeds of Adansonia digitata. Three antibiotics namely Cefotaxime (100–500 ppm), carbenicillen (100–500 ppm), streptomycin (100–500 ppm) were tried to assess their influence on seed germination. Antibiotic stock solutions were added to the autoclaved basal medium ½ Woody Plant medium (WPM) with 0.8 mg l-1 Benzylaminopurine (BAP) and 4% sucrose. Mature seeds without seed coat were usedas explant. The seeds were sterilized using 70% ethanol for 30seconds and 4%sodium hypochlorite for 10 minutes followed by incubation for 30 days in light with photosynthetic photon flux of 6 μmol m-2 s-1. 16 / 8 hour photoperiod. 30 seeds were used for each treatment and the experiment was repeated thrice. 76.6% germination (maximum) was observed in 300 ppm cefataxime treated medium followed by 44.4% at 300 ppm streptomycin and 56.6% at 300 ppm of carbenicillen.The use of streptomycin as an antibiotic did not have a stimulatory effect on seed germination. Percent seed germination was very less as compared to cefataxime and carbenicillen.The study was carried out to develop the most suitable protocol for seed germination in vitro and further regeneration studies on this aesthetically important valuable tree species. However, more variables like the effect of Photoperiod, culture vessels, age of explant etc. on in vitro seed germination have to be standardized to generate a complete protocol.