Mycobacteriophages-derived lysis proteins such as LysB are promising antibiotic alternatives to counter drug-resistant pathogenic mycobacteria such as Mycobacterium tuberculosis and nontuberculous mycobacteria. Previous work has correlated the antimycobacterial activity of LysB with its peptidoglycan-binding domain (PGBD) in the N-terminal domain (NTD), but underlying mechanistic is lacking. Here, we discovered that NTD is crucial for the antimycobacterial activity of TM4LysB due to the likely effect on TM4LysB's translocation to its cognate substrate mycolyl-arabinogalactan-peptidoglycan (mAGP), but neither the effect on NTD's PG-binding properties nor TM4LysB's catalytic activity. We also found that the antimycobacterial activity of several different LysBs, including TM4LysB, D29LysB and Ms6LysB, is strongly influenced by a highly conserved super-secondary structure (SSS) in the catalytic domain which structurally distinguishes LysBs from members of the cutinase family and allows PG-binding ability of catalytic domain. The SSS is essential for both catalytic activity and probably efficient translocation of LysBs to the mAGP layer.