The present study investigates the potential anti-infective and antibacterial properties of phenylalkyl acetophenones and anacardic acids isolated from the ethyl acetate extract of the leaves of Knema oblongifolia, along with synthetic derivatives that were generated. As antibiotic resistance grows, the discovery of new anti-infective agents becomes crucial. The study utilizes a phenotypic screening approach, employing a 3R infection model with Mycobacterium marinum (Mm) and Dictyostelium discoideum (Dd) as proxies for Mycobacterium tuberculosis and human macrophages. This model helps to distinguish between general antibiotics and specific anti-infectives that inhibit bacterial growth inside host cells. A previous screening carried out on a collection of 1600 plant natural extracts revealed K. oblongifolia as a significant source of anti-infective compounds. The ethyl acetate extract of this plant exhibited a strong inhibition of Mm intracellular growth in the infection model while minimally affecting bacterial growth in broth. HPLC bioactivity profiling of this extract based on a high-resolution microfractionation strategy uncovered that the activity was associated with different LC-peaks spread over the chromatogram. LC-MS-based metabolite profiling of the extract revealed that they shared common substructural elements. Based on such information, fractionation of the extract at a larger scale led to the isolation of 12 bioactive natural products (NPs): four newly described acetophenone NPs and eight salicylic acid derivatives (three of which were new). These NPs were further tested for their activities against Mm (antibacterial and anti-infective), Pseudomonas aeruginosa , and Staphylococcus aureus . Additionally, the study involved de novo synthesis of derivatives based on the backbones of the isolated acetophenones to enhance their bioactivity. Hemisynthesis on one of the isolated natural acetophenone was also carried out and resulted in an increase in potency but no increase in selectivity toward the inhibition of Mm intracellular growth. Overall, biological activity assessments revealed that some of the synthetic analogues generated were better candidates in terms of both selectivity and potency, with an improved activity profile compared to natural analogues. The best synthetic candidate reached an IC₅₀ of 0.59 μM for the inhibition of intracellular bacterial growth during infection (anti-infective activity).