This study aimed to analyze the chemotactic response of differentiated HL-60 neutrophil-like (dHL-60) cells to trans-anethole (TA)-treated Staphylococcus aureus strains. Special attention was paid to evaluate the influence of TA on the chp gene expression level, as well as molecular docking and molecular dynamics (MD) simulation studies on interactions of TA with chemotaxis inhibitory protein of S. aureus (CHIPS). The following parameters were studied: susceptibility to TA using the agar diffusion method, the chp gene detection and its expression under TA influence, and clonal diversity of S. aureus strains using molecular techniques. Furthermore, a chemotactic response of dHL-60 cells to TA-treated S. aureus using Boyden chamber assay was detected and molecular modeling using both the docking methodology and unbiased MD simulations was conducted. It was found that TA showed antibacterial activity against all strains. Three genotypes and one unique pattern were distinguished among the strains. 50% of the isolates were chp-positive. It was observed that TA reduced/inhibited chp gene expression in most S. aureus strains. Enhanced chemotactic response of dHL-60 cells to TA-treated S. aureus strains was also noted. This correlation was similar for both chp-positive and chp-negative strains. Both molecular docking and MD simulations studies confirmed that TA is preferentially bound in the complement component 5a/CHIPS interface interaction region and can interfere with any processes exploiting this binding cavity. It has been proven that dHL-60 cells exhibited a higher chemotactic response to TA-treated S. aureus strains in comparison to non-treated bacteria, regardless of the achieved expression of the chp gene or its lack. Nevertheless, further analyses are required to understand this mechanism better.