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It is widely recognized that microbial inoculants (MI) and bio-organic fertilizers (BOFs) containing beneficial microorganisms can play an important role in improving orchard soil properties and enhancing fruit quality. However, insufficient data regarding the relevant fruit quality effects hindered the supplementary MI and BOFs in kiwifruit cultivation. Using conventional fertilization management as the control, this study investigated the impacts of supplementary applications of MI and BOFs at two gradient dosages on the harvest-time quality and cold storage characteristics of ‘Puyu’ yellow-fleshed kiwifruit. Regarding leaf physiological indices and soil pH, MI-3.0 and BOF-20 treatments significantly elevated total chlorophyll content at 60 days after flowering (DAF) (the fruit expansion stage). Leaf nitrogen (N), phosphorus (P) and potassium (K) contents declined gradually during fruit development, while MI-2.0 and BOF-10 treatments markedly promoted leaf P accumulation at 20–100 DAF. Additionally, the MI-2.0 treatment significantly reduced 20–40 cm subsoil pH, which is favorable for kiwifruit plants that prefer acidic and slightly acidic conditions. On the other hand, appropriate doses of MI and BOF treatments exerted a significant effect on improving the quality of kiwifruit at the ripening stage. These effects were mainly manifested in the increased single fruit weight, firmness, dry matter content and total soluble solids (TSSs) of kiwifruit following MI-3.0 and BOF-20 treatments. Furthermore, MI-3.0 and BOF-10 notably elevated the fructose and glucose contents in both flesh and core, as well as sucrose and ascorbic acid (AsA) contents in the flesh; MI-2.0 and BOF treatments significantly increased citric and malic acids in the core and quinic acid in the flesh. During cold storage, the BOF-20 treatment not only delayed the occurrence of the ethylene peak by 20 d and significantly reduced its peak value, but also alleviated the decline in total acid content at the middle storage stage (20–40 d). Additionally, MI-2.0 and BOF-20 treatments effectively delayed kiwifruit softening at the early storage stage (0–10 d), and MI treatments maintained a high AsA content in the core during 10–20 d of cold storage. MI and BOF fertilization treatments had little effect on the dynamic change trends of sucrose synthase (SuS), sucrose phosphate synthase (SPS) and acid invertase (AI) in kiwifruit during cold storage, only exerting significant effects at specific time points. In conclusion, supplementary applications of MI and BOFs could improve kiwifruit quality at the harvest stage by positively regulating the accumulation of dry matter, soluble sugars and organic acid contents, and also have the potential to enhance the storage performance of kiwifruit. These findings provide a scientific basis for establishing an effective fertilization regime for kiwifruit.