In the present study, thin-layer chromatography (TLC) method was adopted for the fractionation of antibacterial substances from the crude extracts obtained in 1:1(v/v) chloroform : methanol of red seaweeds comprising Portieria hornemannii (Lyngbye) P. Silva, Gracilaria crassa (Harvey) J. Ag., brown Dictyota bartyrensiana Lamouroux, Hydroclathrus clathratus (C.Agardh) M.A.Howe, Sargassum wightii Greville, green Ulva (Enteromorpha) compressa (Linnaeus) Nees, and Halimeda macroloba Decaisne collected along the Coast of Pamban (Rameswaram, Gulf of Mannar, India) at four different seasons namely post-monsoon (February), summer (May), pre-monsoon (August) and monsoon (November) seasons for the year 2009. Fractionated substances detected under UV (Mechanical + disinfection) and iodine were tested for antibacterial activity against the plant pathogenic bacterium Xanthomonas axonopodis pv. citri (Hasse) Vauterin et al. which cause canker in citrus. Number of active fractions with high antibacterial activity was recorded from the crude extracts obtained in seaweeds collected during monsoon and post-monsoon whereas seaweeds collected during summer and pre-monsoon showed low antibacterial activity. UV active substances fractionated at R f 0.23 from red seaweed Porteiria hornemannii showed maximum antibacterial activity among the fractionated substances obtained on TLC from the crude extracts of all the seaweeds investigated.