The phytochemical analysis of Gossypium hirsutum was carried out using standardized methods for the evaluation of active compounds and provided opportunities for use in the preparation of new drugs. Bacillus thuringiensis (Bt) and non-Bacillus thuringiensis (non-Bt) cotton species are picked for the current study and during this, different solvents such as acetone, ethanol, methanol, pet-ether, chloroform, water used for phytochemical and antimicrobial screening from cotton leaves and seeds in the year 2013. The phytochemical analysis found the presence and absence of flavonoids, phenols, glycosides, saponins, amino acids and tannins, alkaloids, anthraquinones, steroids, terpenoids, sugars respectively. The investigation on the antibacterial efficacy against pathogenic strains of bacteria has been conducted and the highest inhibitory effect is present in leaf chloroform extract of non-Bt cotton against Pseudomonas fluorescens, whereas leaf methanol extract of Bt cotton was found to be most potent against Escherichia coli. Ethyl acetate and chloroform extracts have been effective against the remaining bacterial strains. Water extracts have a great inhibiting effect on all fungal pathogens tested. Results suggest that some samples may contain various pharmacologically active compounds with moderate antimicrobial properties.