The introduction of a switchable function into the structure of a bioactive compound can endow it with unique capabilities for regulating biological activity under the influence of various types of external stimuli, which makes such hybrid compounds promising objects for photopharmacology, targeted drug delivery and bio-imaging. This work is devoted to the synthesis and study of new spirocyclic derivatives of important human hormones—β-estradiol and estrone—possessing a wide range of biological activities. The obtained hybrid compounds represent an indoline spiropyrans family, a widely known class of organic photochromic compounds. The structure of the compounds was confirmed by ¹H and ¹³C NMR, IR, HRMS and single-crystal X-ray analysis. The intermolecular interactions in the crystals of spiropyran (3) were defined by Hirshfeld surfaces and 2D fingerprint plots, which were successfully acquired from CrystalExplorer (v21.5). All target hybrids demonstrated pronounced activity in the visible region of the spectrum. The mechanisms of thermal isomerization processes of spiropyrans and their protonated merocyanine forms were studied by DFT methods, which revealed the energetic advantage of the protonation process with the formation of a β-cisoid CCCH conformer at the first stage and its further isomerization to more stable β-transoid forms. The proposed mechanism of acidochromic transformation was confirmed by the additional NMR study data that allowed for the detecting of the intermediate CCCH isomer. The study of the short-term cytotoxicity of new spirocyclic derivatives of estrogens and their 2-formyl-precursors was performed on the HeLa cell model. The precursors and spiropyrans differed in toxicity, suggesting their variable applicability in novel anti-cancer technologies.