In the present study, preservatives and antibiotics were applied in a variety of doses to assess their effects on the beer waste-based protein bait, a product widely used to control populations of Bactrocera dorsalis (Diptera: Tephritidae). Screening was performed using the inhibition zone method against the dominant bacteria in deteriorated protein bait. Additionally, the attractiveness of the bait was assessed after respectively adding different doses of methyl eugenol, brown sugar solution, ammonium acetate, and guava essence, as determined by bio-cage tests. The results showed that the four selected preservatives had different inhibition effects at different dose levels, and that the level of inhibition increased with dose: at the highest dose of 1:1 (v:v), sodium benzoate can significantly inhibit Staphylococcus spp., potassium sorbate can significantly inhibit Lactobacillus spp., sodium nitrite can significantly inhibit Escherichia spp. and ethyl p-hydroxybenzoate can significantly inhibit Staphylococcus spp. and Xanthomonas spp. Among three antibiotics, kanamycin was the most effective in suppressing a mix of all four bacteria for all tested doses. In terms of lure performance, the addition of four substances in various ratios into the protein bait were found to have varying effects on its attractiveness to B. dorsalis females and males. Specifically, the addition of a high-dose of guava essence could significantly enhance the attractiveness of the bait. Our findings will help to enhance the effectiveness of protein bait applications by prolonging the quality guarantee period and improving its attractiveness toward both female and male B. dorsalis.