The application of fluorogenic and chromogenic enzymatic substrates in culture media continues to offer huge potential for accessible bacterial detection and identification. However, their clinical use poses two major disadvantages: the presence of commensal and/or coliform bacteria camouflages the detection of bacteria of interest, and false results may arise due to secretion of a similar enzyme by a different species (or strain of) that acts upon the same enzymatic substrates. In the current work, selective antimicrobial agents or suicide substrates were developed and investigated in order to prevent the growth of these interfering bacteria, thus improving the integrity of the detection and diagnosis of common clinical infections. This project has been carried out under collaboration between University of Sunderland (UK), bioMérieux (France) and Freeman Hospital, Newcastle-upon-Tyne (UK). The L-alanine analogues D/L-fosfalin and -chloro-L-alanine were synthesised, both of which target alanine racemase, and different peptide derivatives based on these moieties were prepared using the well-established IBCF/NMM coupling approach with a series of protection/deprotection methods. Two dipeptide derivatives based on para-aminobenzoic acid (PABA) were also investigated. A series of synthetic derivatives were subjected to microbiology evaluation using 20 different clinical strains of clinical bacteria at the Freeman Hospital. The stability of -Cl-L-Ala-D/L-Fos was investigated at pH 6.1 – 9.1; hydrolysis products were formed above pH 6.1. The minimum inhibitory concentration (MIC) of L-Ala-L-Ala-D/L-Fos was double the concentration of L-Ala-L-Ala-L-Fos, showing that only the diastereoisomer with L-fosfalin exhibited significant inhibitory activity against the bacteria. The introduction of -chloro-L-alanine into the phosphonopeptide derivatives enhanced the antibacterial activity; for example the growth of commensal bacterium, Escherichia coli, was inhibited at a low MIC (0.125 – 0.5 mg/L) by most X--Cl-L-Ala-D/L-Fos tripeptide analogues, except when X: sarcosine (MIC: 4 mg/L). However, no significant antibacterial activity was found in the PABA derivatives. These di- and tri-peptide fosfalin-containing derivatives, especially with the inclusion of -Cl-L-alanine, offer improved selectivity for the detection and identification of pathogenic bacteria in clinical samples by restricting the over-growth of commensal bacteria.