Genetic transformation of plants is pivotal for advancing biotechnology, with success depending largely on effective selection methods. Marchantia polymorpha has emerged as a model plant due to its evolutionary importance, ease of manipulation, and simple genetic structure. However, inconsistent antibiotic performance and limited studies on optimal selection agent concentrations have posed challenges. This study aimed to optimize selection agent use in M. polymorpha genetic transformation. We assessed the effects of five antibiotics (hygromycin, kanamycin, G418, neomycin, and gentamicin) and the herbicide chlorsulfuron on M. polymorpha gemmae growth by evaluating survival, morphological properties, area, and fresh weight of the thallus on these selection media. For each agent, we identified the minimum lethal concentration for nontransgenic plants and safe thresholds for transgenics, balancing false-positive prevention with reduced toxicity. This study effectively determined optimal concentrations of selective agents for M. polymorpha gemmae transformation. Hygromycin, G418, and chlorsulfuron have broad selective concentration ranges of 5–100, 2–100 µg/ml, and 20–400 ng/ml, respectively, enabling efficient transformant selection. On the other hand, kanamycin, gentamicin, and neomycin had narrow selection concentration ranges, making the separation of transformed and nontransformed gemmae less effective. Notably, we observed cross-activity of the gentamicin resistance enzyme with G418, a phenomenon also confirmed in tobacco. This unexpected cross-activity underscores the need for careful marker selection while also highlighting its potential for strategic antibiotic use. Specifically, this cross-activity could improve the usability of the aacC1 selection marker, making it a more versatile tool for molecular biological analysis. Our findings enhance transformation protocols for M. polymorpha and possibly other plant species.