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MALDI-MS is now replacing the conventional cumbersome bacterial identification techniques with its high efficiency, reliability and rapidity. One of the methodologies facilitating increased detection sensitivity of bacterial cells by MALDI-MS is through the use of nanoparticle (NP) based affinity probes. The present paper brings out the bifunctional property of Ag NPs in acting as affinity probes at ideal concentrations and bactericidal at higher concentrations. These interactions have been explained on the basis of the studies conducted with two model bacterial systems, Escherichia coli and Serratia marcescens. This work highlights the importance of NP concentration when used for affinity capture of bacteria. The results of the paper indicate a critical concentration specific to every bacteria and hence the need to identify the critical concentration of affinity probes (CCAP) for a specific NP with respect to individual bacterial species before considering its use as affinity probe for bacterial studies. The CCAP for Ag NPs was identified in the present study to be 1 mL L(-1) in the case of E. coli and was 0.5 mL L(-1) for S. marcescens. So far, MALDI-MS results were categorized as qualitative only; in this paper we have used a methodology for converting the mass data to yield quantitative results. The MYSTAT software based Tukey-Kramer multiple comparison tests proved that these threshold values (CCAP) were statistically significant. Above these concentrations Ag NPs were found to show pronounced bactericidal activity rather than affinity probe properties. The following study also demonstrates a modified protocol in that the NPs were incubated with the bacterial cultures at low concentrations, instead of the usual method of adding NPs at high concentrations to well grown bacterial cultures. A possible mechanism for the mode of action of NPs in enhancing bacterial detection sensitivity in the MALDI-MS is also proposed in this paper.