Virulence-associated genes allow plant pathogenic bacteria to adapt effectively to their hosts. Among them, the function of type III effectors (T3Es) has been well documented. T3Es are introduced directly into host cells via the type III secretion system (T3SS). The presence of five Type III effector genes was tested in thirty-four X. citri pv. malvacearum isolates from major cotton-growing regions of India. Thirteen isolates carried the avrBs3 avirulence gene. Eight isolates were positive for the xopK gene, seven for the HopG1 gene, six for the xopAD gene, and five for the xopI gene. Defense gene expression was studied in cotton using PR-1 (chitinase), PR-5 (ribonuclease-like protein), and polyubiquitin as an internal control in a susceptible cotton cultivar (LRA5166) by quantitative real-time PCR. The differential gene expression profiles were studied in cotton leaves by pre-treatment of cotton seedlings with effective biocontrol agents such as Streptomyces mutabilis (RAL2) and Bacillus amyloliquefaciens (ETL2), along with salicylic acid (1000 ppm), before X. citri pv. malvacearum infection. Plants treated with S. mutabilis (RAL2) showed numerically maximum expression of the defense gene PR1 compared to those treated with B. amyloliquefaciens (ETL2). PR5 gene expression was numerically higher in plants treated with B. amyloliquefaciens (ETL2) than in those treated with S. mutabilis (RAL2).