The incidence of Mycobacterium marinum infection is on the rise; however, the existing drug treatment cycle is lengthy and often requires multi-drug combination. Therefore, there is a need to develop new and effective anti-M. marinum drugs. Cochliomycin A, a 14-membered resorcylic acid lactone with an acetonide group at C-5′ and C-6′, exhibits a wide range of antimicrobial, antimalarial, and antifouling activities. To further explore the effect of this structural change at C-5′ and C-6′ on this compound’s activity, we synthesized a series of compounds with a structure similar to that of cochliomycin A, bearing ketal groups at C-5′ and C-6′. The R/S configuration of the diastereoisomer at C-13′ was further determined through an NOE correlation analysis of CH3 or CH at the derivative C-13′ position and the H-5′ and H-6′ by means of a 1D NOE experiment. Further comparative 1H NMR analysis of diastereoisomers showed the difference in the chemical shift (δ) value of the diastereoisomers. The synthetic compounds were screened for their anti-microbial activities in vitro. Compounds 15–24 and 28–35 demonstrated promising activity against M. marinum, with MIC90 values ranging from 70 to 90 μM, closely approaching the MIC90 of isoniazid. The preliminary structure–activity relationships showed that the ketal groups with aromatic rings at C-5′ and C-6′ could enhance the inhibition of M. marinum. Further study demonstrated that compounds 23, 24, 29, and 30 had significant inhibitory effects on M. marinum and addictive effects with isoniazid and rifampicin. Its effective properties make it an important clue for future drug development toward combatting M. marinum resistance.