A 120-membered chalcone library has been designed and prepared from six differently substituted acetophenones (A1-A6) and 20 benzaldehydes (B1-B20). The library was subjected to biological studies targeted against six bacterial strains. For the identification of the most-active member(s) of the library, the so-called indexed or positional-scanning method was applied. Six out of 26 sub-libraries, i.e., AL1-AL6, were synthesized by keeping the acetophenone moiety A fixed and using equimolar quantities of the 20 different benzaldehydes. The remaining 20 sub-libraries BL1-BL20 were prepared by keeping the benzaldehyde B component fixed and varying the six acetophenones (Table 1). The bactericidal activities of the resulting sub-libraries were tested and used as indices to the rows or columns of a two-dimensional matrix. Finally, parallel synthesis of 24 specific members with the highest-expected antibacterial activities, present in two sub-libraries, was carried out. These chalcones were screened again, and the results were exploited for establishing the structure-activity relationship (SAR) and the identification of the lead compound, which turned out to be 1,3-bis(2-hydroxyphenyl)prop-2-en-1-one (A2B2) in terms of activity towards Staphylococcus aureus and Bacillus subtilis (Tables 5-7).