Early diagnosis and therapeutic monitoring of acute osteomyelitis (AO) is challenging. Here, we use a polyethylene glycol (PEG)ylated chemotactic peptide cinnamoyl-F-(D)L-F-(D)L-F (cFLFLF) conjugated with hydrazinonicotinamide (HYNIC) and labeled with Tc-99m ([Tc-99m]cFLFLF) to image AO in a rat model and to validate its efficacy in early diagnosis and therapeutic evaluation of AO. Forty rats were divided into eight groups of five each. Groups A, B, C, G, and H were AO models, and D, E, and F were sham controls. Groups A and D underwent [Tc-99m]cFLFLF scintigraphy, groups B and E underwent [Tc-99m]methylene diphosphonate ([Tc-99m]MDP) bone scan, and groups C and F underwent 2-deoxy-2-[F-18]fluoro-d-glucose ([F-18]FDG) positron emission tomography (PET)/computed tomography (CT) scan. [Tc-99m]cFLFLF biodistribution was assessed in group G. The response to antibiotic therapy was evaluated using [Tc-99m]cFLFLF scintigraphy in group H. Conventional radiographs were obtained following scintigraphy. Ratios of infected or sham-operated tibia to the opposite tibia (T/B) were calculated. Immediately after the imaging studies, infected tibias were excised and underwent histopathological analysis and immunohistochemistry staining. AO was present in all rats of groups A, B, C, G, and H. Total histological scores were not significantly different among groups A, B, and C (F = 0.34, p = 0.71). The biodistribution results revealed significant uptake and excellent retention of [Tc-99m]cFLFLF in the infected tibia. [Tc-99m]cFLFLF scintigraphy and [Tc-99m]MDP bone scan both detected AO. The mean T/B ratio of [Tc-99m]cFLFLF scintigraphy 1 h postinjection was 2.09-fold higher than that of [Tc-99m]MDP bone scan (t = 13.81, p < 0.001). The mean T/B ratio of [F-18]FDG PET/CT scan was not significantly different from the control group F (t = 2.17, p = 0.062). [Tc-99m]cFLFLF scintigraphy revealed a significant attenuation of inflammation in group H following a 3-week antibiotic treatment, which was verified by histopathological analysis and immunohistochemistry staining. Our results suggest that the specificity and image quality of [Tc-99m]cFLFLF are superior to those of the [Tc-99m]MDP and [F-18]DFG imaging probes currently used for early diagnosis of AO. Furthermore, [Tc-99m]cFLFLF was able to effectively evaluate the therapeutic response to antibiotic treatment of AO. Our data suggest that [Tc-99m]cFLFLF is a promising imaging agent for detection of infectious diseases.