The presence of multiple nuclei in a common cytoplasm poses a significant challenge to genetic modification in mushrooms. Here, we demonstrate successful gene editing in both nuclei of a dikaryotic strain of Ganoderma lucidum using the Cas9-gRNA ribonucleoprotein complex (RNP). The RNP targeting the pyrG gene was introduced into dikaryotic protoplasts of G. lucidum, resulting in the isolation of 31 mycelial colonies resistant to 5-fluoroorotic acid (5-FOA). Twenty-six of these isolates were confirmed as dikaryotic strains by the presence of two distinct A mating type markers, denoted as A1 and A2. All dikaryons exhibited clamp connections on their mycelial hyphae, while the remaining 5 transformants were monokaryotic. Subsequent sequence analysis of PCR amplicons targeting pyrG revealed that two dikaryons harbored disrupted pyrG in both nuclei (pyrG-/pyrG-), while 10 and 14 displayed pyrG+/pyrG- (A1/A2) and pyrG-/pyrG+ (A1/A2) configurations, respectively. The disruption was achieved through non-homologous end joining repair, involving deletion or insertion of DNA fragments at the site of the double-strand break induced by RNP. Importantly, the nuclei were stable throughout 10 serial transfers over a period of 6 months. These findings highlight the capability of RNP to target genes across multiple nuclei within the same cytoplasm.