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The white spots known as microbubbles that are formed in cellulose acetate film, significantly affects the texture of the image. However, the formation of microbubbles and their corresponding microstructure are poorly understood. In this paper, cellulose acetate microfilm from the Republic of China (AD 1912–1949) collected in the Second Historical Archives of China was chosen as a prototype to investigate the causes of microbubble disease for the first time. A comparative study was conducted between the film samples with and without microbubbles. Herein, the microstructural analyses including optical and SEM microscopy were conducted. The crystals in the microbubbles proved to be triphenyl phosphate as identified by NMR spectroscopy and SEM–EDX. Comparative properties of the film substrate with and without microbubbles were measured by TG, XRD and FT-IR spectroscopy. The results show that formation of microbubbles was generally accompanied with the vinegar syndrome and was produced between the protective and emulsion layers with the different types of shapes along the closed structure (the diameter of ~ 0.1–2 mm). The formed microbubble was filled with plasticizer. Based on the above-mentioned results, the possible formation process of microbubble was proposed in the present study. The results can provide support for the cause of microbubbles and basis for the conservation of photographic films. [ABSTRACT FROM AUTHOR]